Nucleic acid encoding DS-CAM proteins and products related thereto

ABSTRACT

In accordance with the present invention, there are provided novel Down Syndrome-Cell Adhesion Molecule (DS-CAM) proteins. Nucleic acid sequences encoding such proteins and assays employing same are also disclosed. The invention DS-CAM proteins can be employed in a variety of ways, for example, for the production of anti-DS-CAM antibodies thereto, in therapeutic compositions and methods employing such proteins and/or antibodies. DS-CAM proteins are also useful in bioassays to identify agonists and antagonists thereto.

This is a non-provisional application based on, and claims the benefitof, U.S. Provisional Application No. 60/029,322 filed Oct. 25, 1996, thecontent of which is incorporated herein by reference in its entirety.

ACKNOWLEDGMENT

This invention was made with Government support under Grant NumbersHL50025 and HD17449 awarded by the National Institutes of Health andDE-FG03-92ER61402 awarded by the Department of Energy. The Governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to nucleic acids and proteins encodedthereby. Invention nucleic acids encode a novel N-CAM member of theimmunoglobulin superfamily of proteins. The invention also relates tomethods for making and using such nucleic acids and proteins.

BACKGROUND OF THE INVENTION

Research spanning the last decade has significantly elucidated themolecular events attending cell-cell interactions in the body,especially those events involved in the movement and activation of cellsin the immune system. See generally, Springer et al., Nature346:425-434, 1990. Cell surface proteins, and especially the so-calledCellular Adhesion Molecules (“CAMs”) have correspondingly been thesubject of pharmaceutical research and development having as its goalintervening in the processes of leukocyte extravasation to sites ofinflammation and leukocyte movement to distinct target tissues. Theisolation and characterization of cellular adhesion molecules, thecloning and expression of DNA sequences encoding such molecules, and thedevelopment of therapeutic and diagnostic agents relevant toinflammatory process, viral infection and cancer metastasis have alsobeen the subject of numerous U.S. and foreign applications for LettersPatent. See Edwards, Current Opinion in Therapeutic Patents1(11):1617-1630, 1991 and particularly the published “patent literaturereferences” cited therein.

Numerous CAMs have been characterized to date. See, for example,vascular adhesion molecule (VCAM-1) as described in PCT WO 90/13300;platelet endothelial cell adhesion molecule (PECAM-1)described in Newmanet al., Science 247:1219-1222, 1990; and PCT WO 91/10683; and thefollowing U.S. Pat. Nos. 5,525,487; 5,235,049; 5,272,263; 5,489,233;5,264,554; 5,318,890; 5,389,520; 5,519,008; and the like.

There is substantial evidence that N-CAM and its relatives play animportant part in neural development (Edelman and Crossin, “CELLADHESION MOLECULES: Implications for a Molecular Histology”, Ann. Rev.Biochem. 60:155-190, 1991; and Walsh and Doherty, Curr. Opinion in CellBiol. 5:791-796, 1993). For example, antibodies directed against N-CAMsdisturbed the normal growth pattern of nerve processes. N-CAM (locus11q23.1) is expressed in large amounts in cells of the developing neuraltube, but when neural crest cells dissociate from the neural tube andmigrate away, they lose N-CAM, only to reexpress it later when theyreaggregate to form a neural ganglion. In addition, Rosenthal et al.,(Nature Genet. 2:107-112, 1992) reported that mutations in CAM-L1 (locusXq28) cause X-linked hydrocephalus, and Jouet et al., (Nature Genet.7:402-407, 1994) showed that mutations in CAM L1 gene are responsiblefor type 1 X-linked spastic paraplegia and MASA syndrome which showsagenesis of the corpus callosum. Therefore, there is a need in the artto identify and isolate novel N-CAM members of the immunoglobulinsuperfamily so that their role in neural development and neural cellcommunication can be determined.

Therefore, there continues to be a need in the art for the discovery ofadditional proteins participating in human cell-cell interactions andespecially a need for information serving to specifically identify andcharacterize such proteins in terms of their amino acid sequence.Moreover, to the extent that such molecules might form the basis for thedevelopment of therapeutic and diagnostic agents, it is essential thatthe DNA encoding them be elucidated. The present invention satisfiesthis need and provides related advantages as well.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided isolatednucleic acids encoding novel mammalian N-CAM (neural-cell adhesionmolecule) members of the immunoglobulin superfamily of proteins,referred to herein as Down Syndrome-Cell Adhesion Molecules (DS-CAMs).Further provided are vectors containing invention nucleic acids, probesthat hybridize thereto, host cells transformed therewith, antisenseoligonucleotides thereto and related compositions. The nucleic acidmolecules described herein can be incorporated into a variety ofrecombinant expression systems known to those of skill in the art toreadily produce isolated DS-CAM proteins. In addition, the nucleic acidmolecules of the present invention are useful as probes for assaying forthe presence and/or amount of a DS-CAM gene or mRNA transcript in agiven sample. The nucleic acid molecules described herein, andoligonucleotide fragments thereof, are also useful as primers and/ortemplates in a PCR reaction for amplifying genes encoding DS-CAMproteins.

In accordance with the present invention, there are also providedisolated mammalian DS-CAM proteins. These proteins are useful, forexample, in neural prosthetic devices used in entubulation methods ofrepairing (regenerating) damaged or severed peripheral nerves (see,e.g., U.S. Pat. No. 4,955,892, incorporated herein by reference). Inaddition, these proteins, or fragments thereof, are useful as immunogensfor producing anti-DS-CAM antibodies, or in therapeutic compositionscontaining such proteins and/or antibodies. Invention DS-CAM proteinsare also useful in bioassays to identify agonists and antagoniststhereto. Also provided are transgenic non-human mammals that express theinvention protein.

Antibodies that are immunoreactive with invention DS-CAM proteins arealso provided. These antibodies are useful in diagnostic assays todetermine levels of DS-CAM proteins present in a given sample, e.g.,tissue samples, Western blots, and the like. The antibodies can also beused to purify DS-CAM proteins from crude cell extracts and the like.Moreover, these antibodies are considered therapeutically useful tocounteract or supplement the biological effect of DS-CAMs in vivo.

Methods and diagnostic systems for determining the levels of DS-CAMprotein in various tissue samples are also provided. These diagnosticmethods can be used for monitoring the level of therapeuticallyadministered DS-CAM protein or fragments thereof to facilitate themaintenance of therapeutically effective amounts. These diagnosticmethods can also be used to diagnose physiological disorders that resultfrom abnormal levels or abnormal structures of the DS-CAM protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a physical map of the localization of the DS-CAM gene to aregion between D21S345 and D21S347 on chromosome 21. The locations ofBAC clones (starting with numbers) and PAC clones (starting with “P”)are indicated by horizontal bars. An arrow head indicates a gap in theBAC and PAC contig. The location of the DS-CAM gene is indicated by athick arrow.

FIG. 2 shows the predicted amino acid sequence of the human DS-CAM1protein corresponding to SEQ ID NO:2 and a schematic structure. IG:Immunoglobulin type-C2 domain. FbN: Fibronectin type III domain. Thebold Cs in the amino acid sequence indicates Cysteine residues formingdisulfide bonds in the Ig-like type-C2 domains. The bold NXS and NXT inthe amino acid sequence correspond to potential N-glycosylation sites.

FIG. 3 shows a partial genomic structure of DS-CAM1 and a deletioncontained in DS-CAM2 cDNA clones (clones pDS-CAM-18 and pDS-CAM-52). Thedeletion boundary sequence (GC-AG) suggests an unusual alternativesplicing. The horizontal bar represents genomic sequence containingexons of DS-CAM-42. Exons are indicated by open boxes. Exon-intronboundaries are defined by a comparison of the cDNA sequence ofpDS-CAM-42 and genomic sequence determined from a BAC clone.

FIG. 4 shows a schematic comparison of neuronal Ig superfamily members.Ig-like type C-2 domains, fibronectin type III domains and transmembranedomains are indicated. MAG: myelin-associated glycoprotein, N-CAM:neural cell adhesion molecule, BIG-1: brain-derived immunoglobulin (Ig)superfamily molecule-1, DCC: deleted in colorectal carcinoma.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided isolatednucleic acids, which encode novel mammalian members of the DS-CAM familyof proteins, and fragments thereof. The phrase “DS-CAM” refers tosubstantially pure native DS-CAM protein, or recombinantly producedproteins, including naturally occurring allelic variants thereof encodedby mRNA generated by alternative splicing of a primary transcript, suchas DS-CAM1 (SEQ ID NO:2) and DS-CAM2 (SEQ ID NO:11) disclosed herein,and further including fragments thereof which retain at least one nativebiological activity, such as immunogenicity. In one aspect, inventionDS-CAM proteins, such as DS-CAM1, are cell-surface glycoproteins thatare mobile in the plane of the membrane. Invention DS-CAM1 proteinscontain extra- and intra-cellular domains that transduce informationfrom the outside of the cell to the cytoplasm and the nucleus, therebydetermining cell function. In another aspect, invention DS-CAM proteins,such as DS-CAM2, are non-membrane bound, soluble proteins.

In one aspect of the invention DS-CAM proteins are further characterizedas comprising at least 7 Immunoglobulin-like (Ig-like) domainshomologous to the immunoglobulin superfamily and 6 type III fibronectinrepeats (see, e.g., Edelman and Crossin, “CELL ADHESION MOLECULES:Implications for a Molecular Histology”, Ann. Rev. Biochem., 60:155-190,1991; and Walsh and Doherty, Curr. Opinion in Cell Biol., 5:791-796,1993; each of which is incorporated herein by reference in itsentirety). In another aspect of the invention, DS-CAM proteins are thoseproteins comprising at least 8, preferably at least 9 Ig-like domains,with at least 10 Ig-like domains being especially preferred.

As used herein, “Ig-like domains”, or grammatical variations thereof,refers to the well known repeats that are common among Cell AdhesionMolecules (CAMs) (see, e.g., FIG. 1A at p. 158 of Edelman and Crossin,supra, 1991; and Walsh and Doherty, supra, 1993; each of which isincorporated herein by reference in its entirety).

The phrase “type III fibronectin repeats”, “fibronectin repeats,” orgrammatical variations thereof, refers to the well known repeats thatare common among Cell Adhesion Molecules (CAMs)(see, e.g., FIG. 1A at p.158 of Edelman and Crossin, supra, 1991; and Walsh and Doherty, supra,1993; each of which is incorporated herein by reference in itsentirety).

The invention DS-CAM proteins define a novel sub-class of the Ig(immunoglobulin) superfamily with highest homologies to the neural celladhesion molecules including BIG-1 (Yoshihara et al., Neuron 13:415-426,1994), CAM-L1 (Moos et al., Nature 334:701-703, 1988), DCC (Fearon etal., Science 247:49-56, 1990), neogenin (Lane et al., Genomics35:456-465, 1996), and contactin (Ranscht, J. Cell Bio. 107:1561-1573,1988) (FIG. 4). It has been found that the structure of invention DS-CAMproteins is unique within the neural immunoglobulin superfamily, and isdistinctive due to the number of Ig-like type C2 and fibronectin IIIdomains (10 and 6 respectively) and from the interruption of the fourthand fifth fibronectin domains by a 10th C2 domain, the functionalsignificance of which may be of interest. The novel structure of DS-CAMand its expression throughout the nervous system during differentiationsuggest interesting roles for the neural CAM in neural development andfunction. The location of DS-CAM in a region critical for DSneurocognitive phenotypes provides a human model in which to test thesignificance of these roles for cognitive function.

The neural Ig-superfamily members play critical roles in neuraldevelopment and function and have been implicated in cell migration andsorting, axon guidance and fasciculation, formation of neuralconnections, and in synaptic plasticity (Edelman and Crossin, supra,1991, Walsh and Doherty, supra, 1993; Tessier-Lavigne et al., Science274:1123-1133, 1996: Shuster et al., Neuron 17:641-654, 1996: Shuster etal. Neuron 17:655-657, 1996). These activities are mediated by thehomophilic or heterophilic binding properties of Ig-superfamily members(Mauro et al., J. Cell Bio. 119:191-202, 1992 and Milev et al., J. Biol.Chem. 271:15716-15723, 1996), the binding of Ig-superfamily proteins toextracellular matrix proteins (Grumet et al., Cell Adhesion Comm.1:177-190, 1993; Taira et al., Neuron 12:861-872, 1994; and Zisch etal., J. Cell Bio. 119:203-213, 1992), and the binding to smallerdiffusible chemorepellents or chemoattractants, for example, DCC andnetrin (Keino-Masu et al., Cell 87:175-185, 1996).

The specificity of DS-CAM expression for the central nervous system andthe timing of its expression to the period of neurite outgrowth in boththe central and peripheral nervous systems, indicates a role for DS-CAMin early development and differentiation (Examples 4 and 5). Early indevelopment when, with the exception of neural crest precursors,expression is clearly absent from regions that contain dividingneuroepithelial precursors such as the ependymal layer of the neuraltube and the ventricular zone of the brain (Altman and Bayer, Atlas ofPrenatal Rat Brain Development, CRC Press, Ann Arbor, Mich., 1995). Inthe embryo, differentiated neurons express DS-CAM when they havefinished migrating to their proper positions within the neuroepithelium,during neurite outgrowth.

Neural crest cells may express DS-CAM while they are migrating. At 15.5and 16.5 days pc, most of the neural crest derived tissues have someexpression, although not all have finished migration. The continuedexpression of DS-CAM in the myenteric plexus after 15.5-16.5 dpc is dueto the neural crest cells that have stopped dividing, although othersare in the cell cycle. Approximately 50% of myenteric ganglia neuronsarise after birth and DS-CAM may be expressed later in this subset. Atlater stages, the data suggest that DS-CAM is down regulated in theneural crest derivatives such as the myenteric ganglia and ganglia ofthe pancreas. The DS-CAM expression in tissues derived from the neuralcrest is of interest with respect to the high level detected in theumbilical cord. The tissue surrounding the umbilical artery and vein isderived from the neural crest and functions in coordinating thecardiovascular changes occurring at birth. The expression detected inthe fetal liver and branchial arches is also derived from neural crestrelated to the ductus venosus and ultimately the ductus arteriosus andcardiac outflow tracts, respectively.

DS-CAM expression continues post-natally, in the differentiating regionsof the newborn brain, such as, the septum and inferior colliculus, andin the adult in regions associated with plasticity, such as, theolfactory bulb and hippocampus. When combined with the evidence forinvolvement of the Ig superfamily in determining synaptic strength(Mayford et al., Science 256:638-644, 1992)), the continued expressionsupports a role for DS-CAM in remodeling, learning and memory. Theexpression pattern and the role of dendritic connections in cell bodymaintenance indicate that an increase in DS-CAM expression in DS brainis responsible in part for the abnormalities of dendritic structure anddecreased intersections seen at four months post-natal in DSindividuals.

Alternatively spliced variants of CAMs have distinct roles in differentparts of the brain, as demonstrated for closely related Ig-superfamilymembers, such as, NCAM (Cunningham et al., Science 236:799-806, 1987 andFigarella-Branger et al., J. Neuropathol. Exp. Neurol. 51:12-23, 1992).The differential expression of alternatively spliced DS-CAM transcriptsencoding DS-CAM1 (SEQ ID NO:2) and DS-CAM2 (SEQ ID NO:11) has likewisebeen observed in various parts of the human adult brain. For example, ithas been found that DS-CAM clones encoding DS-CAM2 contain a smalldeletion relative to DS-CAM1, which deletion contains the transmembranedomain (Example 3 and FIG. 3) and results in a stop codon 36 bpdownstream. The results of RT-PCR (Example 5) indicated that all RNAstested from various human tissues expressed both the DS-CAM1 and DS-CAM2transcripts and that the PCR products generated the sequence and sizepredicted for the appropriate form. The proximal and distal borders ofthe deletion are located within neighboring exons and reveal variantconsensus splice site sequences (Jackson, Nuc. Acid Res. 19:3795-3798,1991) with further surrounding homology to the U1 spliceosome RNA.

From Northern analyses (Example 4) a minimum of three distincttranscripts are recognized by a probe for the transmembrane domain. FromcDNA sequence analyses (Example 5) two forms of the DS-CAM protein arededuced, one that generates a transmembrane adhesion molecule and asecond that is deleted for the transmembrane domain, thereby generatinga molecule that is transported to the extracellular matrix. This mode ofgenerating extracellular and membrane bound forms of CAMs is insurprising contrast to the GPI (glycosylphosphatidylinositol) linkageused by most CAMs, and would provide a way of generating longer rangehomophilic interactions between cells and the extracellular matrix,which may be significant for cell migration.

The DS-CAM gene was isolated (as described in the Examples hereinafter)by using the BAC contig on 21q22.2-q22.3 covering the region betweenD21S55 and MX1 (Hubert et al., Genomics 41:218-226, 1997). The genespans a minimum of 900 kb, estimated by summing the size of BACs andPACs that are non-overlapping and covered by the DS-CAM gene (FIG. 1).The DS-CAM gene covers a gap in all physical maps of this region. Fromhybridization experiments indicating no signal of the complete cDNA toBAC 277G10 covering 210 kb, a 5′ intron is at least this size, similarto the first intron of the DCC gene (Cho et al., Genomics 19:525-531,1994). Alternatively, other alternative transcripts can contain exonslocated in this BAC. The gene spans the boundary of bands 21q22.2 andq22.3, a Giemsa-dark and Giemsa-light band, respectively. The locationof the gene for PEP19, a small 634 bp gene with large introns within thesame band 21q22.2 (Cabin et al., Somat. Cell Mol. Genet. 22:167-175,1996) suggests a general structure of genes in G-bands having largeintrons.

The nucleic acid molecules described herein are useful for producinginvention DS-CAM proteins, when such nucleic acids are incorporated intoa variety of protein expression systems known to those of skill in theart. In addition, such nucleic acid molecules or fragments thereof canbe labeled with a readily detectable substituent and used ashybridization probes for assaying for the presence and/or amount of aDS-CAM gene or mRNA transcript in a given sample. The nucleic acidmolecules described herein, and fragments thereof, are also useful asprimers and/or templates in a PCR reaction for amplifying genes encodingthe invention protein described herein.

The term “nucleic acid” (also referred to as polynucleotides)encompasses ribonucleic acid (RNA) or deoxyribonucleic acid (DNA),probes, oligonucleotides, and primers. DNA can be either complementaryDNA (cDNA) or genomic DNA, e.g. a gene encoding a DS-CAM protein. Onemeans of isolating a nucleic acid encoding a DS-CAM polypeptide is toprobe a mammalian genomic library with a natural or artificiallydesigned DNA probe using methods well known in the art. DNA probesderived from the DS-CAM gene are particularly useful for this purpose.DNA and cDNA molecules that encode DS-CAM polypeptides can be used toobtain complementary genomic DNA, cDNA or RNA from mammalian (e.g.,human, mouse, rat, rabbit, pig, and the like), or other animal sources,or to isolate related cDNA or genomic clones by the screening of cDNA orgenomic libraries, by methods described in more detail below. Examplesof nucleic acids are RNA, cDNA, or isolated genomic DNA encoding aDS-CAM polypeptide. Such nucleic acids may include, but are not limitedto, nucleic acids having substantially the same nucleotide sequence asset forth in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, or at least nucleotides 453-6185 set forth in SEQ ID NO:1, ornucleotides 453-5168 set forth in SEQ ID NO:10.

Use of the terms “isolated” and/or “purified” in the presentspecification and claims as a modifier of DNA, RNA, polypeptides orproteins means that the DNA, RNA, polypeptides or proteins so designatedhave been produced in such form by the hand of man, and thus areseparated from their native in vivo cellular environment. As a result ofthis human intervention, the recombinant DNAs, RNAs, polypeptides andproteins of the invention are useful in ways described herein that theDNAs, RNAs, polypeptides or proteins as they naturally occur are not.

As used herein, “mammalian” refers to the variety of species from whichthe invention DS-CAM protein is derived, e.g., human, rat, mouse,rabbit, monkey, baboon, bovine, porcine, ovine, canine, feline, and thelike. A preferred DS-CAM protein herein, is human DS-CAM.

In one embodiment of the present invention, cDNAs encoding the inventionDS-CAM proteins disclosed herein include substantially the samenucleotide sequence as set forth in SEQ ID NO:1, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, or SEQ ID NO:10. Preferred cDNA molecules encodingthe invention proteins include the same nucleotide sequence asnucleotides 453-6185 set forth in SEQ ID NO:1, or nucleotides 453-5168set forth in SEQ ID NO:10.

As employed herein, the term “substantially the same nucleotidesequence” refers to DNA having sufficient identity to the referencepolynucleotide, such that it will hybridize to the reference nucleotideunder moderately stringent hybridization conditions. In one embodiment,DNA having substantially the same nucleotide sequence as the referencenucleotide sequence encodes substantially the same amino acid sequenceas that set forth in SEQ ID NO:2 or SEQ ID NO:11, or the DS-CAM codingregion of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, or a larger aminoacid sequence including SEQ ID NO:2 or SEQ ID NO:11, or the DS-CAMcoding region of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9. In anotherembodiment, DNA having “substantially the same nucleotide sequence” asthe reference nucleotide sequence has at least 60% identity with respectto the reference nucleotide sequence. DNA having at least 70%, morepreferably at least 90%, yet more preferably at least 95%, identity tothe reference nucleotide sequence is preferred.

This invention also encompasses nucleic acids which differ from thenucleic acids shown in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10 but which have the same phenotype. Phenotypicallysimilar nucleic acids are also referred to as “functionally equivalentnucleic acids”. As used herein, the phrase “functionally equivalentnucleic acids” encompasses nucleic acids characterized by slight andnon-consequential sequence variations that will function insubstantially the same manner to produce the same protein product(s) asthe nucleic acids disclosed herein. In particular, functionallyequivalent nucleic acids encode polypeptides that are the same as thosedisclosed herein or that have conservative amino acid variations, orthat encode larger polypeptides that includes SEQ ID NO:2 or SEQ IDNO:11, or the DS-CAM coding region of SEQ ID NO:7, SEQ ID NO:8 or SEQ IDNO:9. For example, conservative variations include substitution of anon-polar residue with another non-polar residue, or substitution of acharged residue with a similarly charged residue. These variationsinclude those recognized by skilled artisans as those that do notsubstantially alter the tertiary structure of the protein.

Further provided are nucleic acids encoding DS-CAM polypeptides that, byvirtue of the degeneracy of the genetic code, do not necessarilyhybridize to the invention nucleic acids under specified hybridizationconditions. Preferred nucleic acids encoding the invention polypeptidesare comprised of nucleotides that encode substantially the same aminoacid sequences set forth in SEQ ID NO:2 or SEQ ID NO:11, or the DS-CAMcoding region of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

Thus, an exemplary nucleic acid encoding an invention DS-CAM protein maybe selected from:

-   -   (a) DNA encoding the amino acid sequence set forth in SEQ ID        NO:2 or SEQ ID NO:11, or the DS-CAM coding region of SEQ ID        NO:7, SEQ ID NO:8 or SEQ ID NO:9,    -   (b) DNA that hybridizes to the DNA of (a) under moderately        stringent conditions, wherein said DNA encodes biologically        active DS-CAM, or    -   (c) DNA degenerate with respect to either (a) or (b) above,        wherein said DNA encodes biologically active DS-CAM.

Hybridization refers to the binding of complementary strands of nucleicacid (i.e., sense:antisense strands or probe:target-DNA) to each otherthrough hydrogen bonds, similar to the bonds that naturally occur inchromosomal DNA. Stringency levels used to hybridize a given probe withtarget-DNA can be readily varied by those of skill in the art.

The phrase “stringent hybridization” is used herein to refer toconditions under which polynucleic acid hybrids are stable. As known tothose of skill in the art, the stability of hybrids is reflected in themelting temperature (T_(m)) of the hybrids. In general, the stability ofa hybrid is a function of sodium ion concentration and temperature.Typically, the hybridization reaction is performed under conditions oflower stringency, followed by washes of varying, but higher, stringency.Reference to hybridization stringency relates to such washingconditions.

As used herein, the phrase “moderately stringent hybridization” refersto conditions that permit target-DNA to bind a complementary nucleicacid that has about 60% identity, preferably about 75% identity, morepreferably about 85% identity to the target DNA; with greater than about90% identity to target-DNA being especially preferred. Preferably,moderately stringent conditions are conditions equivalent tohybridization in 50% formamide, 5× Denhardt's solution, 5×SSPE, 0.2% SDSat 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 65° C.

The phrase “high stringency hybridization” refers to conditions thatpermit hybridization of only those nucleic acid sequences that formstable hybrids in 0.018M NaCl at 65° C. (i.e., if a hybrid is not stablein 0.018M NaCl at 65° C., it will not be stable under high stringencyconditions, as contemplated herein). High stringency conditions can beprovided, for example, by hybridization in 50% formamide, 5× Denhardt'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C.

The phrase “low stringency hybridization” refers to conditionsequivalent to hybridization in 10% formamide, 5× Denhardt's solution,6×SSPE, 0.2% SDS at 42° C., followed by washing in 1×SSPE, 0.2% SDS, at50° C. Denhardt's solution and SSPE (see, e.g., Sambrook et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989) are well known to those of skill in the art as are othersuitable hybridization buffers.

As used herein, the term “degenerate” refers to codons that differ in atleast one nucleotide from a reference nucleic acid, e.g., SEQ ID NO:1,but encode the same amino acids as the reference nucleic acid. Forexample., codons specified by the triplets “UCU”, “UCC”, “UCA”, and“UCG” are degenerate with respect to each other since all four of thesecodons encode the amino acid serine.

Preferred nucleic acids encoding the invention polypeptide(s) hybridizeunder moderately stringent, preferably high stringency, conditions tosubstantially the entire sequence, or in certain embodiments substantialportions (i.e., typically at least 15-30 nucleotides) of the nucleicacid sequence set forth in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9 or SEQ ID NO:10.

The invention nucleic acids can be produced by a variety of methodswell-known in the art, e.g., the methods described herein, employing PCRamplification using oligonucleotide primers from various regions of SEQID NO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and thelike.

In accordance with a further embodiment of the present invention,optionally labeled DS-CAM-encoding cDNAs, or fragments thereof, can beemployed to probe library(ies) (e.g., cDNA, genomic, and the like) foradditional nucleic acid sequences encoding novel mammalian DS-CAMproteins. As described in Example 3, construction of mammalian cDNAlibraries, preferably a human trisomy 21 fetal brain cDNA library, iswell-known in the art. Screening of such a cDNA library is initiallycarried out under low-stringency conditions, which comprise atemperature of less than about 42° C., a formamide concentration of lessthan about 50%, and a moderate to low salt concentration.

Presently preferred probe-based screening conditions comprise atemperature of about 37° C., a formamide concentration of about 20%, anda salt concentration of about 5× standard saline citrate (SSC; 20×SSCcontains 3M sodium chloride, 0.3M sodium citrate, pH 7.0). Suchconditions will allow the identification of sequences which have asubstantial degree of similarity with the probe sequence, withoutrequiring perfect homology. The phrase “substantial similarity” refersto sequences which share at least 50% homology. Preferably,hybridization conditions will be selected which allow the identificationof sequences having at least 70% homology with the probe, whilediscriminating against sequences which have a lower degree of homologywith the probe. As a result, nucleic acids having substantially the samenucleotide sequence as nucleotides 453-6185 set forth in SEQ ID NO:1, ornucleotides 453-5168 set forth in SEQ ID NO:10, SEQ ID NO:7, SEQ IDNO:8, or SEQ ID NO:9 are obtained.

As used herein, a nucleic acid “probe” is single-stranded DNA or RNA, oranalogs thereof, that has a sequence of nucleotides that includes atleast 14, at least 20, at least 50, at least 100, at least 200, at least300, at least 400, or at least 500 contiguous bases that are the same as(or the complement of) any contiguous bases set forth in any of SEQ IDNO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10. Preferredregions from which to construct probes include 5′ and/or 3′ codingregions of SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ IDNO:10. In addition, the entire cDNA encoding region of an inventionDS-CAM protein, or the entire sequence corresponding to SEQ ID NO:1, SEQID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, may be used as aprobe. Probes may be labeled by methods well-known in the art, asdescribed hereinafter, and used in various diagnostic kits.

As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal. Any label or indicating means can be linked to invention nucleicacid probes, expressed proteins, polypeptide fragments, or antibodymolecules. These atoms or molecules can be used alone or in conjunctionwith additional reagents. Such labels are themselves well-known inclinical diagnostic chemistry.

The labeling means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturation to form afluorochrome (dye) that is a useful immunofluorescent tracer. Adescription of immunofluorescent analytic techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis etal., eds., John Wiley & Sons, Ltd., pp. 189-231, 1982, which isincorporated herein by reference.

In one embodiment, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, and the like. In anotherembodiment, radioactive elements are employed labeling agents. Thelinking of a label to a substrate, i.e., labeling of nucleic acidprobes, antibodies, polypeptides, and proteins, is well known in theart. For instance, an invention antibody can be labeled by metabolicincorporation of radiolabeled amino acids provided in the culturemedium. See, for example, Galfre et al., Meth. Enzymol. 73:3-46, 1981.Conventional means of protein conjugation or coupling by activatedfunctional groups are particularly applicable. See, for example,Aurameas et al., Scand. J. Immunol. 8(7):7-23, 1978; Rodwell et al.,Biotech. 3:889-894, 1984; and U.S. Pat. No. 4,493,795.

In accordance with another embodiment of the present invention, thereare provided isolated mammalian DS-CAM proteins (preferably human),polypeptides, and fragments thereof encoded by invention nucleic acid.Preferably, DS-CAM proteins referred to herein, are those polypeptidesspecifically recognized by an antibody that also specifically recognizesa DS-CAM protein including the sequence set forth in SEQ ID NO:2 or SEQID NO:11, or the DS-CAM coding region of SEQ ID NO:7, SEQ. ID NO:8 orSEQ ID NO:9. Invention isolated DS-CAM proteins are free of cellularcomponents and/or contaminants normally associated with a native in vivoenvironment.

The invention DS-CAM proteins are further characterized as beingprimarily expressed in fetal brain and not expressed in fetal lung orfetal liver. For example, the results of Northern analysis (described inExample 4) using human fetal tissues showed that 8.5 kb and 7.6 kbtranscripts are expressed only in fetal brain and not expressed in fetallung, fetal liver and fetal kidney. Northern blot analyses of adulttissues revealed differential expression of three alternativetranscripts of 9.7 kb, 8.5 kb and 7.6 kb in different substructures ofthe brain. The 9.7 kb transcript is highly expressed in the substantianigra, moderately expressed in the amygdala and hippocampus, and lessexpressed in the whole brain. A similar pattern is observed by using aPCR product spanning the 191 bp deletion found in DS-CAM-18 andDS-CAM-52. The placenta shows faint bands, and the sizes are smallerthan those in brain. In skeletal muscle, a faint band (6.5 kb) isdetected.

The results of RT-PCR (Example 5) demonstrated expression of humanDS-CAM mRNA in fetal and adult brain, in fetal kidney, as well as in abreast carcinoma cell line mRNA. Thus, splice variant cDNA transcriptsencoding a DS-CAM family of proteins are clearly contemplated by thepresent invention.

The region of chromosome locus 21q22.2 from which DS-CAM is derived ispart of the candidate region for holoprosencephaly type I (HPE1). Inaddition, some patients with this region hemizygously deleted showabnormalities of the corpus callosum and schizencephaly. Therefore,DS-CAM is contemplated as the gene, which when defective, deleted orpresent as a duplication, is responsible for holoprosencephaly, agenesisof the corpus callosum and/or structural defects of the brain. Inaddition, DS-CAM may also be responsible for several phenotypes of DownSyndrome including mental retardation as well as, more specifically, theabnormal dendritic structure observed in Down Syndrome. Additional rolesfor DS-CAM were further evaluated by database homology searches usingBLAST X/N and TIGR database analyses. Results of these searches indicatethat DS-CAM shows moderate homology to N-CAM-1 (Cunningham et al.,Science, 236:799-806, 1987) and to DCC (Fearon et al., Science,247:49-56, 1990).

Presently preferred DS-CAM proteins of the invention include amino acidsequences that are substantially the same as the protein sequence setforth in SEQ ID NO:2 or SEQ ID NO:11, or the DS-CAM coding region of SEQID NO:7, SEQ ID NO:8 or SEQ ID NO:9, as well as biologically active,modified forms thereof. Those of skill in the art will recognize thatnumerous residues of the above-described sequences can be substitutedwith other, chemically, sterically and/or electronically similarresidues without substantially altering the biological activity of theresulting receptor species. In addition, larger or smaller polypeptidesequences containing substantially the same sequence as SEQ ID NO:2 orSEQ ID NO:11, or the DS-CAM coding region of SEQ ID NO:7, SEQ ID NO:8 orSEQ ID NO:9, therein (e.g., splice variants) are contemplated.

As employed herein, the term “substantially the same amino acidsequence” refers to amino acid sequences having at least about 50%,preferably at least about 60%, more preferably at least about 70%identity with respect to the reference amino acid sequence, andretaining comparable functional and biological activity characteristicof the protein defined by the reference amino acid sequence. In anotherembodiment of the invention, preferred invention proteins having“substantially the same amino acid sequence” will have at least about80%, more preferably 90% amino acid identity with respect to thereference amino acid sequence; with greater than about 95% amino acidsequence identity being especially preferred. It is recognized, however,that polypeptides (or nucleic acids referred to hereinbefore) containingless than the described levels of sequence identity arising as splicevariants or that are modified by conservative amino acid substitutions,or by substitution of degenerate codons are also encompassed within thescope of the present invention.

The term “biologically active” or “functional”, when used herein as amodifier of invention DS-CAM protein(s), or polypeptide fragmentthereof, refers to a polypeptide that exhibits functionalcharacteristics similar to DS-CAM. For example, one biological activityof DS-CAM is the ability to act as an immunogen for the production ofpolyclonal and monoclonal antibodies that bind specifically to DS-CAM.Thus, an invention nucleic acid encoding DS-CAM will encode apolypeptide specifically recognized by an antibody that alsospecifically recognizes the DS-CAM protein including the sequence setforth in SEQ ID NO:2 or SEQ ID NO:11, or the DS-CAM coding region of SEQID NO:7, SEQ ID NO:8 or SEQ ID NO:9. Such activity may be assayed by anymethod known to those of skill in the art. For example, atest-polypeptide encoded by a DS-CAM cDNA can be used to produceantibodies, which are then assayed for their ability to bind to theprotein including the sequence set forth in SEQ ID NO.:2 or SEQ ID.NO:11, or the DS-CAM coding region of SEQ ID NO:7, SEQ ID NO:8 or SEQ IDNO:9. If the antibody binds to the test-polypeptide and the proteinincluding the sequence set forth in SEQ ID NO:2 or SEQ ID NO:11, or theDS-CAM coding region of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 withsubstantially the same affinity, then the polypeptide possesses therequisite biological activity.

The invention DS-CAM proteins can be isolated by a variety of methodswell-known in the art, e.g., the methods described herein, therecombinant expression systems described herein, precipitation, gelfiltration, ion-exchange, reverse-phase and affinity chromatography, andthe like. Other well-known methods are described in Deutscher et al.,Guide to Protein Purification: Methods in Enzymology 182 (AcademicPress, 1990), which is incorporated herein by reference. Alternatively,the isolated polypeptides of the present invention can be obtained usingwell-known recombinant methods as described, for example, in Sambrook etal., supra., 1989).

An example of the means for preparing the invention polypeptide(s) is toexpress nucleic acids encoding the DS-CAM in a suitable host cell, suchas a bacterial cell, a yeast cell, an amphibian cell (i.e., oocyte), ora mammalian cell, using methods well known in the art, and recoveringthe expressed polypeptide, again using well-known methods. Inventionpolypeptides can be isolated directly from cells that have beentransformed with expression vectors as described below herein. Theinvention polypeptide, biologically active fragments, and functionalequivalents thereof can also be produced by chemical synthesis. Forexample, synthetic polypeptides can be produced using AppliedBiosystems, Inc. Model 430A or 431A automatic peptide synthesizer(Foster City, Calif.) employing the chemistry provided by themanufacturer.

The present invention also provides compositions containing anacceptable carrier and any of an isolated, purified DS-CAM polypeptide,an active fragment thereof, or a purified, mature protein and activefragments thereof, alone or in combination with each other. Thesepolypeptides or proteins can be recombinantly derived, chemicallysynthesized or purified from native sources. As used herein, the term“acceptable carrier” encompasses any of the standard pharmaceuticalcarriers, such as phosphate buffered saline solution, water andemulsions such as an oil/water or water/oil emulsion, and various typesof wetting agents.

Also provided are antisense oligonucleotides having a sequence capableof binding specifically with any portion of an mRNA that encodes DS-CAMpolypeptides so as to prevent translation of the mRNA. The antisenseoligonucleotide may have a sequence capable of binding specifically withany portion of the sequence of the cDNA encoding DS-CAM polypeptides. Asused herein, the phrase “binding specifically” encompasses the abilityof a nucleic acid sequence to recognize a complementary nucleic acidsequence and to form double-helical segments therewith via the formationof hydrogen bonds between the complementary base pairs. An example of anantisense oligonucleotide is an antisense oligonucleotide comprisingchemical analogs of nucleotides.

Compositions comprising an amount of the antisense oligonucleotide,described above, effective to reduce expression of DS-CAM polypeptidesby passing through a cell membrane and binding specifically with mRNAencoding DS-CAM polypeptides so as to prevent translation and anacceptable hydrophobic carrier capable of passing through a cellmembrane are also provided herein. Suitable hydrophobic carriers aredescribed, for example, in U.S. Pat. Nos. 5,334,761; 4,889,953;4,897,355, and the like. The acceptable hydrophobic carrier capable ofpassing through cell membranes may also comprise a structure which bindsto a receptor specific for a selected cell type and is thereby taken upby cells of the selected cell type. The structure may be part of aprotein known to bind to a cell-type specific receptor.

Antisense oligonucleotide compositions are useful to inhibit translationof mRNA encoding invention polypeptides. Synthetic oligonucleotides, orother antisense chemical structures are designed to bind to mRNAencoding DS-CAM polypeptides and inhibit translation of mRNA and areuseful as compositions to inhibit expression of DS-CAM associated genesin a tissue sample or in a subject.

In accordance with another embodiment of the invention, kits fordetecting mutations, duplications, deletions, rearrangements andaneuploidies in chromosome 21 at locus q22.2 comprising at least oneinvention probe or antisense nucleotide.

The present invention provides means to modulate levels of expression ofDS-CAM polypeptides by employing synthetic antisense oligonucleotidecompositions (hereinafter SAOC) which inhibit translation of mRNAencoding these polypeptides. Synthetic oligonucleotides, or otherantisense chemical structures designed to recognize and selectively bindto mRNA, are constructed to be complementary to portions of the DS-CAMcoding strand or nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10. The SAOC is designed to bestable in the blood stream for administration to a subject by injection,or in laboratory cell culture conditions. The SAOC is designed to becapable of passing through the cell membrane in order to enter thecytoplasm of the cell by virtue of physical and chemical properties ofthe SAOC which render it capable of passing through cell membranes, forexample, by designing small, hydrophobic SAOC chemical structures, or byvirtue of specific transport systems in the cell which recognize andtransport the SAOC into the cell. In addition, the SAOC can be designedfor administration only to certain selected cell populations bytargeting the SAOC to be recognized by specific cellular uptakemechanisms which bind and take up the SAOC only within select cellpopulations.

For example, the SAOC may be designed to bind to a receptor found onlyin a certain cell type, as discussed supra. The SAOC is also designed torecognize and selectively bind to target mRNA sequence, which maycorrespond to a sequence contained within the sequence shown in SEQ IDNO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10. The SAOC isdesigned to inactivate target mRNA sequence by either binding theretoand inducing degradation of the mRNA by, for example, RNase I digestion,or inhibiting translation of mRNA target sequence by interfering withthe binding of translation-regulating factors or ribosomes, or inclusionof other chemical structures, such as ribozyme sequences or reactivechemical groups which either degrade or chemically modify the targetmRNA. SAOCs have been shown to be capable of such properties whendirected against mRNA targets (see Cohen et al., TIBS 10:435, 1989 andWeintraub, Sci. American January 1990, pp. 40; both incorporated hereinby reference).

In accordance with yet another embodiment of the present invention,there is provided a method for the recombinant production of inventionDS-CAM protein(s) by expressing the above-described nucleic acidsequences in suitable host cells. Recombinant DNA expression systemsthat are suitable to produce DS-CAM proteins described herein arewell-known in the art. For example, the above-described nucleotidesequences can be incorporated into vectors for further manipulation. Asused herein, vector (or plasmid) refers to discrete elements that areused to introduce heterologous DNA into cells for either expression orreplication thereof.

Suitable expression vectors are well-known in the art, and includevectors capable of expressing DNA operatively linked to a regulatorysequence, such as a promoter region that is capable of regulatingexpression of such DNA. Thus, an expression vector refers to arecombinant DNA or RNA construct, such as a plasmid, a phage,recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the inserted DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome.

As used herein, a promoter region refers to a segment of DNA thatcontrols transcription of DNA to which it is operatively linked. Thepromoter region includes specific sequences that are sufficient for RNApolymerase recognition, binding and transcription initiation. Inaddition, the promoter region includes sequences that modulate thisrecognition, binding and transcription initiation activity of RNApolymerase. These sequences may be cis acting or may be responsive totrans acting factors. Promoters, depending upon the nature of theregulation, may be constitutive or regulated. Exemplary promoterscontemplated for use in the practice of the present invention includethe SV40 early promoter, the cytomegalovirus (CMV) promoter, the mousemammary tumor virus (MMTV) steroid-inducible promoter, Moloney murineleukemia virus (MMLV) promoter, and the like.

As used herein, the term “operatively linked” refers to the functionalrelationship of DNA with regulatory and effector nucleotide sequences,such as promoters, enhancers, transcriptional and translational stopsites, and other signal sequences. For example, operative linkage of DNAto a promoter refers to the physical and functional relationship betweenthe DNA and the promoter such that the transcription of such DNA isinitiated from the promoter by an RNA polymerase that specificallyrecognizes, binds to and transcribes the DNA.

As used herein, expression refers to the process well-known to those ofskill in the art by which polynucleic acids are transcribed into mRNAand translated into peptides or proteins and, optionally thereafter,modified post-translationally. If the invention nucleic acid is derivedfrom genomic DNA, expression may, if an appropriate eukaryotic host cellor organism is selected, include splicing of the mRNA.

Prokaryotic transformation vectors are well-known in the art and includepBluescript and phage Lambda ZAP vectors (STRATAGENE, San Diego,Calif.), and the like. Other suitable vectors and promoters aredisclosed in detail in U.S. Pat. No. 4,798,885, issued Jan. 17, 1989,the disclosure of which is incorporated herein by reference in itsentirety.

Other suitable vectors for transformation of E. coli cells include thepET expression vectors (Novagen, see U.S Pat. No. 4,952,496), e.g.,pET11a, which contains the T7 promoter, T7 terminator, the inducible E.coli lac operator, and the lac repressor gene; and pET 12a-c, whichcontain the T7 promoter, T7 terminator, and the E. coli ompT secretionsignal. Another suitable vector is the pIN-IIIompA2 (see Duffaud et al.,Meth. in Enzymology, 153:492-507, 1987), which contains the lpppromoter, the lacUV5 promoter operator, the ompA secretion signal, andthe lac repressor gene.

Exemplary, eukaryotic transformation vectors, include the cloned bovinepapilloma virus genome, the cloned genomes of the murine retroviruses,and eukaryotic cassettes, such as the pSV-2 gpt system (described byMulligan and Berg, Nature 277:108-114, 1979) the Okayama-Berg cloningsystem (Mol. Cell Biol. 2:161-170, 1982), and the expression cloningvector described by Genetics Institute (Science 228:810-815, 1985), areavailable which provide substantial assurance of at least someexpression of the protein of interest in the transformed eukaryotic cellline.

Particularly preferred base vectors which contain regulatory elementsthat can be linked to the invention DS-CAM-encoding DNAs fortransfection of mammalian cells are cytomegalovirus (CMV) promoter-basedvectors such as pcDNA1 (Invitrogen, San Diego, Calif.), MMTVpromoter-based vectors such as pMAMNeo (Clontech, Palo Alto, Calif.) andpMSG (Pharmacia, Piscataway, N.J.), and SV40 promoter-based vectors suchas pSVβ (Clontech, Palo Alto, Calif.).

In accordance with another embodiment of the present invention, thereare provided “recombinant cells” containing the nucleic acid molecules(i.e., DNA or mRNA) of the present invention. Methods of transformingsuitable host cells, preferably bacterial cells, and more preferably E.coli cells, as well as methods applicable for culturing said cellscontaining a gene encoding a heterologous protein, are generally knownin the art. See, for example, Sambrook et al., supra, 1989.

Exemplary methods of transformation include, e.g., transformationemploying plasmids, viral, or bacterial phage vectors, transfection,electroporation, lipofection, and the like. The heterologous DNA canoptionally include sequences which allow for its extrachromosomalmaintenance, or said heterologous DNA can be caused to integrate intothe genome of the host (as an alternative means to ensure stablemaintenance in the host).

Host organisms contemplated for use in the practice of the presentinvention include those organisms in which recombinant production ofheterologous proteins has been carried out. Exemplary cells forintroducing DNA include cells of mammalian origin (e.g., COS cells,mouse L cells, Chinese hamster ovary (CHO) cells, human embryonic kidney(HEK) cells, African green monkey cells and other such cells known tothose of skill in the art), amphibian cells (e.g., Xenopus laevisoöcytes), yeast cells (e.g., Saccharomyces cerevisiae, Candidatropicalis, Hansenula polymorpha and P. pastoris; see, e.g., U.S. Pat.Nos. 4,882,279, 4,837,148, 4,929,555 and 4,855,231), bacteria (e.g., E.coli), and the like.

In one embodiment, nucleic acids encoding the invention DS-CAM proteinscan be delivered into mammalian cells, either in vivo or in vitro usingsuitable viral vectors well-known in the art. Suitable retroviralvectors, designed specifically for in vivo “gene therapy” methods, aredescribed, for example, in WIPO publications WO 9205266 and WO 9214829,which provide a description of methods for efficiently introducingnucleic acids into human cells in vivo. In addition, where it isdesirable to limit or reduce the in vivo expression of the inventionDS-CAM, the introduction of the antisense strand of the inventionnucleic acid is contemplated.

In accordance with yet another embodiment of the present invention,there are provided anti-DS-CAM antibodies having specific reactivitywith DS-CAM polypeptides of the present invention. Active fragments ofantibodies are encompassed within the definition of “antibody”.Invention antibodies can be produced by methods known in the art usinginvention polypeptides, proteins or portions thereof as antigens. Forexample, polyclonal and monoclonal antibodies can be produced by methodswell known in the art, as described, for example, in Harlow and Lane,Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, 1988),which is incorporated herein by reference. Invention polypeptides can beused as immunogens in generating such antibodies. Alternatively,synthetic peptides can be prepared (using commercially availablesynthesizers) and used as immunogens. Amino acid sequences can beanalyzed by methods well known in the art to determine whether theyencode hydrophobic or hydrophilic domains of the correspondingpolypeptide. Altered antibodies such as chimeric, humanized, CDR-graftedor bifunctional antibodies can also be produced by methods well known inthe art. Such antibodies can also be produced by hybridoma, chemicalsynthesis or recombinant methods described, for example, in Sambrook etal., supra, 1989; and Harlow and Lane, supra, 1988. Both anti-peptideand anti-fusion protein antibodies can be used. (see, for example,Bahouth et al., Trends Pharmacol. Sci. 12:338 1991; Ausubel et al.,Current Protocols in Molecular Biology (John Wiley and Sons, NY 1989)which are incorporated herein by reference).

Antibody so produced can be used, inter alia, in diagnostic methods andsystems to detect the level of DS-CAM protein present in a mammalian,preferably human, body sample, such as tissue or vascular fluid. Suchantibodies can also be used for the immunoaffinity or affinitychromatography purification of the invention DS-CAM protein. Inaddition, methods are contemplated herein for detecting the presence ofDS-CAM polypeptides on the surface of a cell comprising contacting thecell with an antibody that specifically binds to DS-CAM polypeptides,under conditions permitting binding of the antibody to the polypeptides,detecting the presence of the antibody bound to the cell, and therebydetecting the presence of invention polypeptides on the surface of thecell. With respect to the detection of such polypeptides, the antibodiescan be used for in vitro diagnostic or in vivo imaging methods.

Immunological procedures useful for in vitro detection of target DS-CAMpolypeptides in a sample include immunoassays that employ a detectableantibody. Such immunoassays include, for example, ELISA, Pandexmicrofluorimetric assay, agglutination assays, flow cytometry, serumdiagnostic assays and immunohistochemical staining procedures which arewell known in the art. An antibody can be made detectable by variousmeans well known in the art. For example, a detectable marker can bedirectly or indirectly attached to the antibody. Useful markers include,for example, radionucleotides, enzymes, fluorogens, chromogens andchemiluminescent labels.

Invention anti-DS-CAM antibodies are contemplated for use herein tomodulate the activity of the DS-CAM polypeptide in living animals, inhumans, or in biological tissues or fluids isolated therefrom.Accordingly, compositions comprising a carrier and an amount of anantibody having specificity for DS-CAM polypeptides effective to blocknaturally occurring ligands or other DS-CAM-binding proteins frombinding to invention DS-CAM polypeptides are contemplated herein. Forexample, a monoclonal antibody directed to an epitope of DS-CAMpolypeptide molecules present on the surface of a cell and having anamino acid sequence substantially the same as an amino acid sequence fora cell surface epitope of a DS-CAM polypeptide including the amino acidsequence shown in SEQ ID NO:2 or SEQ ID NO:11, or the DS-CAM codingregion of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, can be useful forthis purpose.

The present invention further provides transgenic non-human mammals thatare capable of expressing exogenous nucleic acids encoding DS-CAMpolypeptides. As employed herein, the phrase “exogenous nucleic acid”refers to nucleic acid sequence which is not native to the host, orwhich is present in the host in other than its native environment (e.g.,as part of a genetically engineered DNA construct).

Also provided are transgenic non-human mammals capable of expressingnucleic acids encoding DS-CAM polypeptides so mutated as to be incapableof normal activity, i.e., do not express native DS-CAM. The presentinvention also provides transgenic non-human mammals having a genomecomprising antisense nucleic acids complementary to nucleic acidsencoding DS-CAM polypeptides, placed so as to be transcribed intoantisense mRNA complementary to mRNA encoding DS-CAM polypeptides, whichhybridizes to the mRNA and, thereby, reduces the translation thereof.The nucleic acid may additionally comprise an inducible promoter and/ortissue specific regulatory elements, so that expression can be induced,or restricted to specific cell types. Examples of nucleic acids are DNAor cDNA having a coding sequence substantially the same as the codingsequence shown in SEQ ID NO:1. An example of a non-human transgenicmammal is a transgenic mouse. Examples of tissue specificity-determiningelements are the metallothionein promoter and the L7 promoter.

Animal model systems which elucidate the physiological and behavioralroles of DS-CAM polypeptides are also provided, and are produced bycreating transgenic animals in which the expression of the DS-CAMpolypeptide is altered using a variety of techniques. Examples of suchtechniques include the insertion of normal or mutant versions of nucleicacids encoding a DS-CAM polypeptide by microinjection, retroviralinfection or other means well known to those skilled in the art, intoappropriate fertilized embryos to produce a transgenic animal. See, forexample, Hogan et al., Manipulating the Mouse Embryo: A LaboratoryManual (Cold Spring Harbor Laboratory, 1986).

Also contemplated herein, is the use of homologous recombination ofmutant or normal versions of DS-CAM genes with the native gene locus intransgenic animals, to alter the regulation of expression or thestructure of DS-CAM polypeptides (see, Capecchi et al., Science244:1288, 1989; Zimmer et al., Nature 338:150, 1989; which areincorporated herein by reference). Homologous recombination techniquesare well known in the art. Homologous recombination replaces the native(endogenous) gene with a recombinant or mutated gene to produce ananimal that cannot express native (endogenous) protein but can express,for example, a mutated protein which results in altered expression ofDS-CAM polypeptides.

In contrast to homologous recombination, microinjection adds genes tothe host genome, without removing host genes. Microinjection can producea transgenic animal that is capable of expressing both endogenous andexogenous DS-CAM protein. Inducible promoters can be linked to thecoding region of nucleic acids to provide a means to regulate expressionof the transgene. Tissue specific regulatory elements can be linked tothe coding region to permit tissue-specific expression of the transgene.Transgenic animal model systems are useful for in vivo screening ofcompounds for identification of specific ligands, i.e., agonists andantagonists, which activate or inhibit protein responses.

Invention nucleic acids, oligonucleotides (including antisense), vectorscontaining same, transformed host cells, polypeptides and combinationsthereof, as well as antibodies of the present invention, can be used toscreen compounds in vitro to determine whether a compound functions as apotential agonist or antagonist to invention polypeptides. These invitro screening assays provide information regarding the function andactivity of invention polypeptides, which can lead to the identificationand design of compounds that are capable of specific interaction withone or more types of polypeptides, peptides or proteins.

In accordance with still another embodiment of the present invention,there is provided a method for identifying compounds which bind toDS-CAM polypeptides. The invention proteins may be employed in acompetitive binding assay. Such an assay can accommodate the rapidscreening of a large number of compounds to determine which compounds,if any, are capable of binding to DS-CAM proteins. Subsequently, moredetailed assays can be carried out with those compounds found to bind,to further determine whether such compounds act as modulators, agonistsor antagonists of invention proteins.

Another application of the binding assay of the invention is the assayof test samples (e.g., biological fluids) for the presence or absence ofDS-CAM. Thus, for example, serum from a patient displaying symptomsthought to be related to over- or under-production of DS-CAM can beassayed to determine if the observed symptoms are indeed caused by over-or under-production of DS-CAM.

In another embodiment of the invention, there is provided a bioassay foridentifying compounds which modulate the activity of invention DS-CAMpolypeptides. According to this method, invention polypeptides arecontacted with an “unknown” or test substance (in the presence of areporter gene construct when antagonist activity is tested), theactivity of the polypeptide is monitored subsequent to the contact withthe “unknown” or test substance, and those substances which cause thereporter gene construct to be expressed are identified as functionalligands for DS-CAM polypeptides.

In accordance with another embodiment of the present invention,transformed host cells that recombinantly express invention polypeptidescan be contacted with a test compound, and the modulating effect(s)thereof can then be evaluated by comparing the DS-CAM-mediated response(e.g., via reporter gene expression) in the presence and absence of testcompound, or by comparing the response of test cells or control cells(i.e., cells that do not express DS-CAM polypeptides), to the presenceof the compound.

As used herein, a compound or a signal that “modulates the activity” ofinvention polypeptides refers to a compound or a signal that alters theactivity of DS-CAM polypeptides so that the activity of the inventionpolypeptide is different in the presence of the compound or signal thanin the absence of the compound or signal. In particular, such compoundsor signals include agonists and antagonists. An agonist encompasses acompound or a signal that activates DS-CAM protein expression.Alternatively, an antagonist includes a compound or signal thatinterferes with DS-CAM protein expression. Typically, the effect of anantagonist is observed as a blocking of agonist-induced proteinactivation. Antagonists include competitive and non-competitiveantagonists. A competitive antagonist (or competitive blocker) interactswith or near the site specific for agonist binding. A non-competitiveantagonist or blocker inactivates the function of the polypeptide byinteracting with a site other than the agonist interaction site.

As understood by those of skill in the art, assay methods foridentifying compounds that modulate DS-CAM activity generally requirecomparison to a control. One type of a “control” is a cell or culturethat is treated substantially the same as the test cell or test cultureexposed to the compound, with the distinction that the “control” cell orculture is not exposed to the compound. For example, in methods that usevoltage clamp electrophysiological procedures, the same cell can betested in the presence or absence of compound, by merely changing theexternal solution bathing the cell. Another type of “control” cell orculture may be a cell or culture that is identical to the transfectedcells, with the exception that the “control” cell or culture do notexpress native proteins. Accordingly, the response of the transfectedcell to compound is compared to the response (or lack thereof) of the“control” cell or culture to the same compound under the same reactionconditions.

Since it is well-known that CAMs interact with extracellular ligands, itis contemplated that invention DS-CAM proteins interact withextracellular ligands. In another embodiment of the present invention,it is contemplated that invention DS-CAM proteins act specifically inconcert or in competition with other CAMs. Thus, the present inventioncontemplates various bioassays for identifying ligands for inventionDS-CAM proteins. In addition, the present invention contemplates anassay measuring the effect of co-expressing during development eithernormal or defective invention DS-CAMs with other CAMs known in the artto assess the resulting phenotype.

In one embodiment of the present invention, there is provided a bioassayfor evaluating whether test compounds are capable of acting as agonistscomprises:

-   -   (a) culturing cells containing:        -   DNA which expresses DS-CAM protein(s) or functional modified            forms thereof, and        -   DNA encoding a reporter protein, wherein said DNA is            operatively linked to a DS-CAM responsive transcription            element;    -   wherein said culturing is carried out in the presence of at        least one compound whose ability to induce signal transduction        activity of DS-CAM protein is sought to be determined, and        thereafter    -   (b) monitoring said cells for expression of said reporter        protein.

In another embodiment of the present invention, the bioassay forevaluating whether test compounds are capable of acting as antagonistsfor DS-CAM protein(s) of the invention, or functional modified forms ofsaid DS-CAM protein(s), comprises:

-   -   (a) culturing cells containing:        -   DNA which expresses DS-CAM protein(s), or functional            modified forms thereof, and        -   DNA encoding a reporter protein, wherein said DNA is            operatively linked to a DS-CAM responsive transcription            element    -   wherein said culturing is carried out in the presence of:        -   increasing concentrations of at least one compound whose            ability to inhibit signal transduction activity of DS-CAM            protein(s) is sought to be determined, and        -   a fixed concentration of at least one agonist for DS-CAM            protein(s), or functional modified forms thereof; and            thereafter    -   (b) monitoring in said cells the level of expression of said        reporter protein as a function of the concentration of said        compound, thereby indicating the ability of said compound to        inhibit signal transduction activity.    -   In step (a) of the above-described antagonist bioassay,        culturing may also be carried out in the presence of:        -   fixed concentrations of at least one compound whose ability            to inhibit signal transduction activity of DS-CAM protein(s)            is sought to be determined, and        -   an increasing concentration of at least one agonist for            DS-CAM protein(s), or functional modified forms thereof.

In yet another embodiment of the present invention, it is contemplatedthat invention DS-CAM proteins mediate signal transduction through themodulation of adenylate cyclase. For example, when a DS-CAM ligand bindsto DS-CAM, adenylate cyclase causes an elevation in the level ofintracellular cAMP. Accordingly, in one embodiment of the presentinvention, the bioassay for evaluating whether test compounds arecapable of acting as agonists or antagonists comprises:

-   -   (a) culturing cells containing:        -   DNA which expresses DS-CAM protein(s) or functional modified            forms thereof, wherein said culturing is carried out in the            presence of at least one compound whose ability to modulate            signal transduction activity of DS-CAM protein is sought to            be determined, and thereafter    -   (b) monitoring said cells for either an increase or decrease in        the level of intracellular cAMP.

Methods well-known in the art that measure intracellular levels of cAMP,or measure cyclase activity, can be employed in binding assays describedherein to identify agonists and antagonists of the DS-CAM. For example,because activation of some CAMs results in decreases or increases incAMP, assays that measure intracellular cAMP levels can be used toevaluate recombinant DS-CAMs expressed in mammalian host cells.

As used herein, “ability to modulate signal transduction activity ofDS-CAM protein” refers to a compound that has the ability to eitherinduce (agonist) or inhibit (antagonist) signal transduction activity ofthe DS-CAM protein.

Each of the invention bioassays (e.g., those described herein, and thelike), can be conducted as competitive assays by co-expressing one ormore members of the CAM immunoglobulin superfamily of proteins known inthe art, such as N-CAMs, along with invention DS-CAMs. In addition, oneor more members of the CAM immunoglobulin superfamily of proteins knownin the art can be co-expressed with invention DS-CAMs to evaluate theagonistic or antagonistic effect on signal transduction of thenon-DS-CAM members acting in concert with invention DS-CAMS.

In yet another embodiment of the present invention, the activation ofDS-CAM polypeptides can be modulated by contacting the polypeptides withan effective amount of at least one compound identified by theabove-described bioassays.

Members of the N-CAM superfamily of immunoglobulins have previously beenimplicated in disease. For example, various alterations of N-CAM levelshave been seen in degenerative disease, developmental defects, and toxicconditions. Increases in the levels of N-CAM in the cerebrospinal fluidof patients with multiple sclerosis have been observed to parallel theirclinical improvement (Massaro et al., Ital. J. Neurol. Sci. Suppl.6:85-88, 1987). Levels of N-CAM were reported to be elevated in theamniotic fluid of mothers carrying fetuses with neural tube defects(Ibsen et al., J. Neurochem. 41:363-366, 1983). Since many such defectsare likely to be due to mechanical aberrations rather than geneticdefects, confirmation of these results would provide a new diagnosticcomponent for prenatal testing. Another provocative finding relates toobservations on the stimulation of Golgi sialyltransferases by lead(Breen and Regan, Development 104:147-154, 1988; and Cookman et al., J.Neurochem. 49:399-403, 1987). Exposure to lead chloride markedlystimulated sialyltransferase activity from postnatal days 16 to 30 inrate. This time is coincident with the period when N-CAM normallybecomes less sialylated. Thus exposure to lead at critical developmentalperiods would presumably lead to more highly sialylated, less adhesive,forms of N-CAM: this prevention of E-A conversion could have significanteffects on neural development. E-A conversion itself has been found tobe delayed in the mouse mutant staggerer (Edelman and Chuong, Proc.Natl. Acad. Sci. USA, 79:7036-7042, 1982) in conjunction with theconnectivity changes associated with the mutation.

The location and expression of DS-CAM in the Down Syndrome (DS)phenotype is supported by the studies of patients with partial trisomy21. A subset of the DS features, including the typical facial appearanceand mental retardation, were suggested by duplication of band 21q22 only(Niebuhr, Humangenetik 21:99-101, 1974). Other studies mapped thosefeatures and congenital heart disease to the region 21q22.2-q22.3 andbetween D21S2.67 and MX1/MX2 (Korenberg et al., Am. J. Hum. Genet.50:294-302, 1992 and Korenberg et al., Proc. Natl. Acad. Sci. USA91:4997-5001, 1994), a region of about 4 Mb that contains DS-CAM. TheTs65Dn mouse model of DS contains the region of MMU16 (Pgk1-ps1 toMX1/2) that includes DS-CAM and reveals some of the neurobehaviourialfeatures of DS (Reeves et al., Nature Genet. 11:177-183, 1995 andHoltzman et al., Proc. Natl. Acad. Sci. USA 93:13333-13338, 1996).

Close to 6% of DS individuals have Hirschsprung's disease (HSCR) (Garveret al., Clin. Genet. 28:503-5-8, 1985) and more than 10% of all HSCR isassociated with DS (Passarge, New Eng. J. Med. 276:138-143, 1967). Amodifier region of HSCR on chromosome 21q22 (D21S259-D21S156) has beenreported in non-DS HSCR (Puffenberger et al., Hum. Mol. Genet.3:1217-1225, 1994). The DS-CAM gene maps within this small region. Theexpression of DS-CAM in the neural crest derived enteric plexus of thegut was detected by mouse tissue in situ hybridization (Example 7). Thefunction of the DS-CAM protein as a neural cell adhesion molecule andthe association of this region of chromosome 21 with HSCR, indicate thatDS-CAM can play a role in the migration of the cranial neural crest thatpopulate this region. Thus, DS-CAM overexpression is responsible for thechromosome 21 association in non-DS HSCR and for the HSCR seen in DS.

Mutations in the molecule CAM-L1, a molecule more similar to DS-CAM thanto N-CAM (FIG. 4), have established roles in human disease. The resultin X-linked hydrocephalus (Rosenthal et al., Nature Genet. 2:107-112,1992), type 1 X-linked spastic paraplegia and the MASA syndrome(including mental retardation, aphasia, shuffling gait, adducted thumband agenesis of the corpus callosum) (Jouet et al., Nature Genet.7:402-407, 1994). The perturbation of development by the aneuploidexpression of CAM-L1 supports a role for the aneuploid expression ofDS-CAM in the causation of developmental and neurological abnormalities.

In accordance with another embodiment of the present invention, thereare provided methods for diagnosing DS-CAM associated disease, such asmental retardation, holoprosencephaly, agenesis of the corpus callosum,or schizencephaly, said method comprising:

-   -   detecting, in said subject, a genomic or transcribed mRNA        sequence including SEQ ID NO:1 or SEQ ID NO:10, or fragments        thereof.        Preferably, the DS-CAM nucleic acids detected in accordance with        the invention diagnostic methods are either mutated in one form        or another (such as point mutations, deletions, and the like),        or are overexpressed relative to levels of DS-CAM expression in        healthy non-diseased individuals.

In accordance with another embodiment of the present invention, thereare provided diagnostic systems, preferably in kit form, comprising atleast one invention nucleic acid in a suitable packaging material. Thediagnostic nucleic acids are derived from the DS-CAM-encoding nucleicacids described herein. In one embodiment, for example, the diagnosticnucleic acids are derived from SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9 or SEQ ID NO:10. Invention diagnostic systems are useful forassaying for the presence or absence of nucleic acid encoding DS-CAM ineither genomic DNA or in transcribed nucleic acid (such as mRNA or cDNA)encoding DS-CAM.

A suitable diagnostic system includes at least one invention nucleicacid, preferably two or more invention nucleic acids, as a separatelypackaged chemical reagent(s) in an amount sufficient for at least oneassay. Instructions for use of the packaged reagent are also typicallyincluded. Those of skill in the art can readily incorporate inventionnucleic probes and/or primers into kit form in combination withappropriate buffers and solutions for the practice of the inventionmethods as described herein.

As employed herein, the phrase “packaging material” refers to one ormore physical structures used to house the contents of the kit, such asinvention nucleic acid probes or primers, and the like. The packagingmaterial is constructed by well known methods, preferably to provide asterile, contaminant-free environment. The packaging material has alabel which indicates that the invention nucleic acids can be used fordetecting a particular sequence encoding DS-CAM including the nucleotidesequence set forth in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9or SEQ ID NO:10, thereby diagnosing the presence of, or a predispositionfor, holoprosencephaly, agenesis of the corpus callosum, or for severalphenotypes of Down Syndrome including mental retardation, and the like.In addition, the packaging material contains instructions indicating howthe materials within the kit are employed both to detect a particularsequence and diagnose the presence of, or a predisposition for,holoprosencephaly, agenesis of the corpus callosum, or for severalphenotypes of Down syndrome including mental retardation, and the like.

The packaging materials employed herein in relation to diagnosticsystems are those customarily utilized in nucleic acid-based diagnosticsystems. As used herein, the term “package” refers to a solid matrix ormaterial such as glass, plastic, paper, foil, and the like, capable ofholding within fixed limits an isolated nucleic acid, oligonucleotide,or primer of the present invention. Thus, for example, a package can bea glass vial used to contain milligram quantities of a contemplatednucleic acid, oligonucleotide or primer, or it can be a microtiter platewell to which microgram quantities of a contemplated nucleic acid probehave been operatively affixed.

“Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter, such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions, and the like.

All U.S. patents and all publications mentioned herein are incorporatedin their entirety by reference thereto. The invention will now bedescribed in greater detail by reference to the following non-limitingexamples.

Materials and Methods

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA, 1982; Sambrook et al., supra,1989; Davis et al., Basic Methods in Molecular Biology, Elsevier SciencePublishing, Inc., New York, USA, 1986; or Methods in Enzymology: Guideto Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. KimmerlEds., Academic Press Inc., San Diego, USA, 1987.

Libraries.

Construction of Bacterial Artificial Chromosome (BAC) library. BAClibrary construction of total human genomic DNA was performed asdescribed in Shizuya et al., Proc. Natl. Acad. Sci. USA 89:8794-8797,1992; and Hubert et al., Genomics 41:218-226, 1997. Yeast artificialchromosome (YAC) clones were obtained from the CEPH mega-YAC library andgrown under standard conditions (Cohen et al., Nature 366:689-701 1993).

P1 artificial chromosome (PAC) library construction. A 3X human PAClibrary, designated RPCI-1 (Ioannou et al., Hum. Genet. 219-220, 1994)was constructed as described (Ioannou et al., Nat. Genet. 6:84-89,1994). The library was arrayed in 384 well dishes. Subsequently, STSsgenerated by sequencing of clones using vector primers were used ashybridization probes to gridded colony filters of the PAC library.

YAC DNA preparation. YAC clones were grown in selective media, pelletedand resuspended in 3 ml 0.9 M sorbitol, 0.1M EDTA pH 7.5, then incubatedwith 100 U of lytocase (Sigma, St. Louis, Mo.) at 37° C. for 1 hour.After centrifugation for 5 minutes at 5,000 rpm pellets were resuspendedin 3 ml 50 mM Tris pH 7.45, 20 mM EDTA 0.3ml 10% SDS was added and themixture was incubated at 65° C. for 30 minutes. One ml of 5 M potassiumacetate was added and tubes were left on ice for 1 hour, thencentrifuged at 10,000 rpm for 10 minutes. Supernatant was precipitatedin 2 volumes of ethanol and pelleted at 6,000 rpm for 15 minutes.Pellets were resuspended in TE, treated with RNase and reextracted withphenol-chloroform.

Analysis by fluorescence in situ hybridization (FISH). PAC or BAC cloneswere biotinylated by nicktranslation in the presence of biotin-14-dATPusing the BioNick Labeling Kit (Gibco-BRL). FISH was performedessentially as described (Korenberg et al., Cytogenet. Cell Genet.69:196-200, 1995). Briefly, 400 ng of probe DNA was mixed with 8 ng ofhuman Cot 1 DNA (Gibco-BRL) and 2 μg of sonicated salmon sperm DNA inorder to suppress possible background produced from repetitive humansequences as well as yeast sequences in the probe. The probes weredenatured at 75° C., preannealed at 37° C. for one hour, and applied todenatured chromosome slides prepared from normal male lymphocytes(Korenberg et al., supra, 1995). Post-hybridization washes wereperformed at 40° C. in 2×SSC/50% formamide followed by washes in 1×SSCat 50° C. Hybridized DNAs were detected with avidin-conjugatedfluorescent isothiocyanate (Vector Laboratories). One amplification wasperformed by using biotinylated anti-avidin. For distinguishingchromosome subbands precisely, a reverse banding technique was used,which was achieved by chromomycin A3 and distamycin A double staining(Korenberg et al., supra, 1995). The 35 color images were captured byusing a Photometrics Cooled-CCD camera and BDS image analysis software(Oncor Imaging, Inc.).

Southern blot analysis. Gel electrophoresis of DNA was carried out on0.8% agarose gels in 1×TBE. Transfer of nucleic acids to Nybond N+ nylonmembrane (Amersham) was performed according to the manufacturer'sinstruction. Probes were labeled using RadPrime Labeling System (BRL).Hybridization was carried out at 42° C. for 16 hours in 50% formamide,5×SSPE, 5× Denhardt's 0.1% SDS, 100 mg/ml denatured salmon sperm DNA.The filters were washed once in 1×SSC, 0.1% SDS at room temperature for20 minutes, and twice in 0.1×SSC, 0.1% SDS for 20 minutes at 65° C. Theblots were exposed onto X-ray film (Kodak, X-OMAT-AR).

Sequencing of PAC and BAC endclones. PAC clones were inoculated into 500ml of LB/kanamycin and grown overnight. BAC clones were inoculated into500 ml of LB/chloramphenicol and grown overnight. DNAs were isolatedusing QIAGEN columns according to the vendors protocol with oneadditional phenol/chloroform/isoamylalcohol extraction followed by oneadditional chloroform/isoamylalcohol extraction. Clones were sequencedusing the Gibco-BRL cycle sequencing kit with standard T7 and SP6primers.

EXAMPLE 1 Construction of BAC Contig

To provide stable clones for gene isolation and sequencing initiativesin the D21S55 to MX1 region, contigs were constructed using BacterialArtificial Chromosomes (BACs) and P1 Artificial Chromosomes (PACs). BAClibrary construction of total human genomic DNA was performed asdescribed (Shiyuza et al., supra, 1992; Kim et al., Genomics 34:213-218,1996). A BAC library was screened using several YACs spanning theregion; a PAC library (Iannou et al., Nature Genet. 6:84-89, 1994) wasscreened using radiolabeled STS PCR products and whole BACs in gapfilling initiatives.

The location of these BAC and PAC clones was confirmed by fluorescencein situ hybridization (FISH). Clone to clone Southerns using 24 new STSs(generated from direct sequencing of BAC and PAC ends) along with 35pre-existing STSs were used to show overlaps between BACs and PACs. TheSTS density over the intervals covered in BACs and PACs was 1 STS every60 kb, and 79% of the clones were positive for 2 or more STSs.Approximately 3.5 Mb of the 4-5 Mb D21S55 to MX1 interval is covered in85 BACs and 25 PACs representing 4-fold coverage within the contigs(Hubert et al., Genomics 41:218-226, 1997). The minimal contig sizes asdetermined by counting only non-overlapping clones are: 1100 kb, 900 kb,510 kb, 380 kb and 270 kb. Insert size of BAC clones was measured byrunning pulse-field gel electrophoresis after digesting DNA with NotI.

EXAMPLE 2 Direct cDNA Selection

A modified direct cDNA selection technique (Yamakawa et al., Hum. Mol.Genet. 4:709-716, 1995; Yamakawa et al., Cytogenet. Cell Genet.74:140-145, 1996) was applied to BAC-423A5, BAC-430F1, BAC-628H2,BAC-371H8 and PAC-31P10 (FIG. 1) by using cDNA from trisomy 21 humanfetal brain, and the selected fragments were then subcloned into aplasmid vector.

Total RNA was isolated from 14 week trisomy 21 fetal brain using TRIregion™ (Molecular Research Center, Inc.). Poly (A)⁺ RNA was isolatedusing Poly (A) Quick® mRNA isolation kit (STRATAGENE). Double strandedcDNA was synthesized using SuperScript™ Choice System (GIBCO BRL) from 5μg trisomy 21 fetal brain poly (A)⁺ RNA using 1 μg oligo (dT)₁₅ or 0.1μg random hexamer. The entire synthesis reaction was purified by GeneClean® II kit (BIO101, Inc.) and then kinased. Sau3AI linker wasattached to the cDNA which was subsequently digested with Sau3AI. Thereaction was purified using Gene Clean. MboI linker was attached to thecDNA and the reaction purified by Gene Clean (Morgan et al., supra,1992). The synthesized product was amplified by PCR using one strand ofMboI linker (5′CCTGATGCTCGAGTGAATTC3′) (SEQ ID NO:4) as a primer. PCRcycling conditions were 40 cycles of 94° C./15 seconds, 60° C./23seconds, 72° C./2 minutes in a 100 μl of 1×PCR buffer (Promega), 3 mMMgCl₂, 5.0 units of Taq polymerase (Promega), 2 μM primer and 0.2 mMdNTPs.

Nineteen BAC DNAs (total 2.5 μg) and 2 PAC DNAs between the region ETS2and MX1 were prepared using QIAGEN plasmid kit and were biotinylatedusing Nick Translation Kit and biotin-16-dUTP (Boehringer Manneheim). 3μg of heat denatured PCR amplified cDNA was annealed with 3 μg of heatdenatured COT1 DNA (BRL) in 100 μl hybridization buffer (750 mM NaCl, 50mM NaPO₄ (pH7.2), 5 mM EDTA, 5× Denhardt's, 0.05% SDS and 50% formamide)at 42° C. for two hours. After prehybridization, 1.2 μg of heatdenatured biotinylated BAC DNA was added and incubated at 42° C. for 16hours. cDNA-BAC DNA hybrids were precipitated with EtOH and dissolved in60 μl of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA. After addition of 40 μl 5 MNaCl, the DNA was incubated with magnetic beads (Dynabeads M-280, Dynal)at 25° C. for 1 hour with gentle rotating to allow attachment of the DNAto the magnetic beads. The beads were then washed twice by pipetting in400 μl of 2×SSC, setting in magnet holder (MPC-E_(TM), Dynal) for 30seconds and removing the supernatant. Four additional washes wereperformed in 0.2×SSC at 68° C. for 10 minutes each with transfer of thebeads to new tubes at each wash. cDNAs were eluted in 100 μl ofdistilled water for 10 minutes at 80° C. with occasional mixing. Theeluted cDNAs were amplified by PCR as described above. After twicerepeating the selection procedure using magnetic beads, amplified cDNAswere digested with EcoRI and subcloned into pBlueScript KS+(STRATAGENE). Insert DNAs were isolated from the subclones, and wereanalyzed by Southern hybridization and DNA sequencing.

The direct cDNA selection procedure using 19 BACs and 2 PACs betweenETS2 and MX1 generated a total of 145 unique cDNA fragments. Genbank andTIGR homology searches using FASTA revealed matches to ETS2, HMG14,PEP19, a Na K ATPase, Titan ESTs, MX1 region ESTs, and 14 ESTs ofunknown function. A cDNA library from a trisomy 21 fetal brain at 14weeks gestation was screened using one of these unique cDNA fragmentslabeled “E51” (SEQ ID NO:3).

EXAMPLE 3

Isolation of Human DS-CAM cDNA Using cDNA Library Screening

A trisomy 21 human fetal brain (14 weeks of age) cDNA library wasconstructed using ZAP-cDNA® synthesis kit (STRATAGENE) which generates aunidirectional cDNA library. Briefly, double- stranded cDNA wassynthesized from 5 μg trisomy 21 fetal brain poly(A)⁺ RNA using a hybridoligo(dT)-XhoI linker primer with 5-methyl dCTP. An EcoRI linker wasattached to the cDNA which was subsequently digested with EcoRI andXhoI, and then cloned into UNI-ZAP XR vector (STRATAGENE). The librarywas packaged using Gigapack® II Gold packaging extract. The titer of theoriginal library was 1.1×10⁶ p.f.u./package. The library was amplifiedonce. A blue-white color assay indicated that 99% of the clones hadinserts.

Screening of the trisomy 21 fetal brain cDNA library was performed usingone of the 145 unique cDNA fragments labeled “E51” (SEQ ID NO:3)prepared as described above. Phages were plated to an average density of1×10⁵ per 175 cm² plate. Plaque lifts of 20 plates (2×10⁶ phages) weremade using duplicated nylon membranes (Hybond-N+; Amersham). Hybridizedmembranes were washed to final stringency of 0.2×SSC, 0.1×SDS at 65° C.The filters were exposed overnight onto X-ray film.

Identification of 62 clones were made out of 2×10⁶ clones in theoriginal library. Eighteen of these positive phage clones were convertedto plasmids, and their DNAs were isolated. These cDNAs wereindependently numbered as separate DS-CAM (Down Syndrome Cell AdhesionMolecule) clones. The length of the inserts of these clones ranged from2.4 kb to 6.6 kb. Exon trapping (Buckler et al., Proc. Natl. Acad. Sci.USA 88:4005-4009, 1991; Church et al., Nature Genet. 6:98-105, 1994) wasalso used to isolate cDNAs in the BAC and PAC contig. With thisapproach, three exons identified from BAC-539E7 and one from BAC-430F1were found to identify the same sequences as those isolated by cDNAselection.

Sequence analysis of one of the clones, labeled DS-CAM-42, revealed a6110 bp DNA sequence which contained a large ORF (5687 bp) as well as3′-UTR sequence (423 bp), but the 5′UTR and start codon were not locatedin clone DS-CAM-42. To characterize the 5′ end, two further clones,DS-CAM-18 of 6.5 kb and DS-CAM-52 of 6.6 kb were characterized. Sequenceanalyses of these clones close to the 5′ end overlap with sequence atthe 5′ end of DS-CAM-42. However, DS-CAM-18 extends 416 bp farther 5′,and DS-CAM-52 extends 494 bp farther 5′ than DS-CAM-42. The extra 494 bpsequence extends the ORF by 43 bp at the 5′ end and contains a startcodon. Two stop codons occur 330 bp and 427 bp upstream of the startcodon. The 494 bp of additional 5′ sequence found in DS-CAM-52 combinedwith DS-CAM-42 (6604 bp) yield a consensus cDNA that encodes one isoformof the invention protein labeled DS-CAM1. The DS-CAM1 cDNA contains anopen reading frame of 5730 bp (SEQ ID NO:1) coding for a 1910 amino acidprotein (SEQ ID NO:2; approximately 211 kilodaltons), flanked by 452 bpof 5′-UTR and 422 bp of 3′-UTR. The 5′-UTR is highly GC rich (81% GCover 452 bp) and contains 13 MspI sites, as well as 72 CG and 93 GCdinucleotide pairs.

The DS-CAM1 protein contains an extracellular component at theN-terminus consisting of nine tandemly repeated Ig-like C2 type domainsand a tenth Ig-like C2 domain located between domains four and five ofan array of six repeated fibronectin type III domains (FIG. 2). EachIg-like C2 domain consists of approximately 100 amino acids with a pairof conserved cysteines separated by 49-56 residues. A singletransmembrane domain of 22 amino acids was defined by using the TMBASEprogram (Hoffmann and Stoffel, Biol. Chem. Hoppe-Seyler 374:166, 1993).The remaining 294 amino acids at the C-terminus corresponding to thecytoplasmic domain have partial homologies to the mouse M-phase inducerphosphatase 2 (Kakizuka et al., Genes Dev. 6:578-590, 1992) in tworegions, one with 34% identity and 52% similarity over 46 bp and asecond with 38% identity and 52% similarity over 21 bp. The homolog ofDrosophila glass gene (O'Neill et al., Proc. Natl. Acad. Sci. USA92:6557-6561, 1995) with 30% identity and 52% similarity over 42 bp, andthe mouse delta opioid receptor (Evans et al., Science 258:1952-1955,1992) with 43% identity and 60% similarity over 30 bp. The putativeprotein contains 16 potential N-glycosylation sites.

A homology search of the predicted amino acid sequence of the 5730 bpopen reading frame of DS-CAM1 (SEQ ID NO:1) to genes registered in theGenbank and the EMBL databases was conducted by using the BLAST-Pprogram (Altschul et al., J. Mol. Biol. 215:403-410, 1990). Thepredicted amino acid sequence revealed homologies to multiple proteins(FIG. 4) including CAM-L1 (Moos et al., Nature 334:701-703, 1988), BIG-1(brain-derived immunoglobulin (Ig) superfamily molecule-1) (Yoshihara etal., Neuron 13:415-426, 1994), DCC (deleted in colon cancer) (Fearon etal., Science 247:49-56, 1990), and revealed DS-CAM as defining a novelclass of the immunoglobulin (Ig) superfamily. Homology searches withsequences of Ig type-C2 domains and fibronectin type-III domains of themost highly related Ig-superfamily members (CAM-L1, DCC, and axonin-1)were conducted by using the FASTA program (Pearson and Lipman, Proc.Natl. Acad. Sci. USA 85:2444-2448, 1988).

In addition, a splice variant cDNA sequence encoding a non-membranebound isoform of DS-CAM1, referred to herein as DS-CAM2, is providedherein. Two human DS-CAM cDNA clones (DS-CAM-18 and DS-CAM-52) werefound to contain identical deletions of 191 bp that occur in neighboringexons and that delete bp 5133 to 5323 of the SEQ ID NO:1 cDNA sequenceencoding DS-CAM1 (FIG. 3). The resulting splice variant transcriptencoding DS-CAM2 (SEQ ID NO:10) is deleted for the entire transmembranedomain that is encoded by the more 3′ of these exons. Further, thedeletion changes the reading frame and creates a stop codon 36 bpdownstream of the deletion resulting in a soluble extracellular proteinof 1571 amino acids (SEQ ID NO:11). The distal border of the resultingdeletion contains the canonical AG of the RNA splicing consensusacceptor site. The proximal border contains a variant of the donorsplice site consensus sequence (Jackson, Nucl. Acids Res. 19:3795-3798,1991).

To confirm that the DS-CAM cDNA originated from the BACs and PACs in theDown syndrome region and to determine the genomic size of DS-CAM, thelongest DS-CAM cDNA clones (DS-CAM-42; 6.1 kb, DS-CAM-18; 6.5 kb,DS-CAM-52; 6.6 kb) were hybridized to Southern blots containing the BACand PAC clone contig (FIG. 1). DS-CAM-42, 18 and 52 hybridized to BACs423A5, 430F1, 628H2, 539E7, 371H8, 825E1, 593D1, 261F12, 30E4, 385B7,388F4, and to PACs 31P10, 58D10. BACs 816F6, 116E8, 720G4, 619H8 wereonly positive for DS-CAM-18 and DS-CAM-52 but negative for DS-CAM-42.All other BACs shown in FIG. 1 were negative. These results indicatethat the DS-CAM gene spans 900 kb-1200 kb genomic DNA and covers a gapin this BAC and PAC contig indicated by an arrowhead as well as in theavailable YAC contigs (Korenberg et al., Genome Res. 5:427-443, 1995;Gardiner et al., Somat. Cell Mol. Genet. 21:399-414, 1995). DS-CAM cDNAsequences were confirmed to originate from these BACs and PACs by directsequencing of the BACs and PACs as templates using cDNAsequence-specific primers.

The map position of DS-CAM on chromosome 21q22.2-22.3 was confirmed byusing clone DS-CAM-42 as a probe for fluorescence in-situ hybridization.Two independent experiments were performed and over 100 metaphase cellswere evaluated. Signals were clearly seen on two chromatids of at leastone chromosome in 85% of cells. There were no other double signal sitesseen in greater than 1% of cells.

EXAMPLE 4 Northern Blot Analysis Of Human DS-CAM Expression

Inserts containing DS-CAM cDNA were excised from the base vector bydigestion with XhoI and EcoRI. After labeling using the random primingmethod (RadPrime Labeling System; GIBCO BRL), followed by purificationusing G-50 Sephadex columns (Quick Spin Column; Boehringer Mannheim),the fragments were used a probes for Northern hybridization usingMultiple Tissue Northern Blot (Clontech). A Northern blot assay wasconducted using DS-CAM cDNA as a probe in various fetal and adulttissues including heart, brain, placenta, lung, liver, skeletal muscle,kidney, and pancreas. Northern hybridization was performed by followingthe manufacturer's instructions. The hybridized membrane was washed at afinal stringency of 0.1×SSC and 0.1×SDS at 50° C. The filter was exposedto X-ray film (Kodak X-OMAT AR) at −70° C. for 1-5 days.

The results of Northern analysis using human fetal tissues showed that8.5 kb and 7.6 kb transcripts are expressed only in fetal brain and notexpressed in fetal lung, fetal liver and fetal kidney. In adult tissues,three transcripts of 9.7 kb, 8.5 kb, and 7.6 kb are present in thebrain. Placenta shows faint bands, and the sizes are similar to those inbrain. In skeletal muscle, a faint smaller band (6.5 kb) is detected. Inmultiple parts of the adult human brain, transcripts of 9.7 kb, 8.5 kband 7.6 kb are differentially expressed. The 9.7 kb transcript is highlyexpressed in the substantia nigra, moderately expressed in amygdala andhippocampus, and less expressed in the whole brain. A similar pattern isobtained using a PCR product which spans the 191 bp deletion found inclones DS-CAM-18 and DS-CAM-52 encoding the splice variant sequencecorresponding to DS-CAM2. Thus, splice variant cDNA transcripts encodinga DS-CAM family of proteins are clearly contemplated by the presentinvention.

EXAMPLE 5 RT-PCR Assays of Human DS-CAM Expression

Reverse-transcriptase polymerase chain reaction (RT-PCR) assays versescDNA libraries of various human tissues were conducted using primersnumbered B9-131F (SEQ ID NO:5) and B9-131R (SEQ ID NO:6). The resultsdemonstrated expression of human DS-CAM mRNA in fetal and adult brain,and fetal kidney. In addition, a breast carcinoma cell line showedexpression of human DS-CAM mRNA.

The cDNAs from 13 independent human fetal and adult sources wereanalyzed by PCR using primer pairs that flanked the alternativelyspliced region that results in a 191 base pair deletion of nucleotides5133-5323 of the DS-CAM1 cDNA set forth in SEQ ID NO:1. The primers weredesigned to generate products of different sizes for each of the twoalternatively spliced transcripts: 536 bp corresponding to thenon-deleted DS-CAM-1 transcript and 345 bp corresponding to the deletedDS-CAM2 transcripts. The analyses included adult samples from amygdala(24 years), skeletal muscle (36 years) and three independentlymphoblastoid cell lines. Fetal samples included whole brain of atrisomy 21 fetus (14 weeks), four from whole brain (4.5-13 weeks), onefrom temporal lobe (28 weeks) and two from heart (4.5 and 13 weeks). Theresults indicate that all fetal and adult samples produced two bandscorresponding to PCR products of the predicted sizes which indicates theexpression of two alternatively spliced transcripts.

EXAMPLE 6 Isolation of Mouse DS-CAM cDNA Clones

A mouse brain cDNA library was prepared from 19 week old female C57Black/6 mice in the Uni-ZAP XR Vector (STRATAGENE). The cDNAs wereoligo-dT primed and cloned unidirectionally into the EcoRI and XhoIsites of the vector. The average insert size is 1.0 kb. The library wasscreened using a human DS-CAM cDNA clone as a probe. Two partial mouseDS-CAM cDNA clones were isolated and sequenced. The combined nucleotidesequences of these clones are set forth in SEQ ID NO:7, SEQ ID NO:8 andSEQ ID NO:9, and were found to represent the 5′, middle and 3′ portions,respectively, of cDNA encoding a mouse DS-CAM.

EXAMPLE 7 Hybridization Analysis of DS-CAM cDNA in Mouse Tissues

BALB/c and C57BL/6×DBA/2 embryos, fetuses and postnatal brains werefixed and embedded as described in detail in Lyons et al., (J. Neurosci.15:5727-5738, 1995). Embryos were fixed in 4% paraformaldehyde inphosphate buffered saline (PBS) overnight, dehydrated and infiltratedwith paraffin. Five to seven micron serial sections were mounted ongelatinized slides. Two sections were mounted/slide, deparaffinized inxylene, rehydrated and post-fixed. The sections were digested withproteinase K, post-fixed, treated with tri-ethanolamine/aceticanhydride, washed and dehydrated. cRNA probes were prepared fromDS-CAM-M-14. The plasmid was linearized with XbaI and T7 polymerase wasused to generate the antisense cRNA. The plasmid was linearized withKpnI and T3 polymerase was used to generate the sense control cRNA. ThecRNA transcripts were synthesized according to manufacturer's conditions(STRATAGENE) and labeled with ³⁵S-UTP (>1000 Ci/mmol; Amersham). cRNAtranscripts larger than 100 nucleotides were subjected to alkalihydrolysis to give a mean size of 70 bases for efficient hybridization.

Sections were hybridized overnight at 52° C. in 50% deionized formamide,0.3M NaCl, 20 mM Tris-HCl pH 7.4, 5 mM EDTA, 10 mM NaPO4, 10% dextransulfate, 1× Denhardt's, 50 μg/ml total yeast RNA, and 50-75,000 cpm/μl³⁵S-labeled cRNA probe. The tissue was subjected to stringent washing at65° C. in 50% formamide, 2×SSC, 10 mM DTT and washed in PBS beforetreatment with 20 μg/ml RNase A at 37° C. for 30 minutes. Followingwashes in 2×SSC and 0.1×SSC for 10 minutes at 37° C., the slides weredehydrated and dipped in Kodak NTB-2 nuclear track emulsion and exposedfor 2-3 weeks in light-tight boxes with desiccant at 4° C. Photographicdevelopment was carried out in Kodak D-19. Slides were counterstainedlightly with toluidine blue and analyzed using both light- and darkfieldoptics of a Zeiss Axiophot microscope. Sense control cRNA probes(identical to the mRNAs) always gave background levels of hybridizationsignal. Embryonic structures were identified with the help of thefollowing atlases: Rugh (The Mouse: Its Reproduction and Development.Oxford Univ. Press, Oxford, UK, 1990), Kaufman (The Atlas of MouseDevelopment. Acad. Press, New York, N.Y., 1992), and Altman and Bayer(supra, 1995).

Tissue in situ hybridization analysis was performed using a mouse cDNAas a probe on sections of normal mouse embryos from days 8.5-17.5 postcoitum (pc) as well as in newborn, two weeks and adult brains asdescribed above. The results indicate that there is no detectableexpression of DS-CAM at 8.5 days pc. At 9.5 days pc, expression wasdetected in the neuroepithelium. Low levels of expression were detectedwithin the branchial arches, suggestive of migrating neural crest cells.At 10.5 days pc, the trigeminal ganglia (neural crest derived) begin toexpress the transcript and expression within the branchial arches wasmore evident.

Expression at 11.5 days pc was abundant throughout the brain. Thetranscript was found within the regions of the nervous system thatdifferentiate earliest during development (Altman and Bayer, supra,1995). In the brain, this includes the ventral-most regions, such as thethalamus and medulla. Some expression was detected within the olfactoryepithelium. Expression within the neural tube begins in two areas: theventrolateral (corresponding to the areas in which the motor neuronsdifferentiate) and the lateral gray columns (that later form commissuralneurons) (Leber et al., J Neurosci. 15:1236-1248, 1990). The dorsal rootganglia (neural crest derived) expressed the transcript at 11.5 days pc.The trigeminal ganglia show higher levels at 11.5 days pc than they didat 10.5 days. Migrating neural crest can be seen within the maxilla, themandibular arch, and in the developing gut. Signal was observed withinthe mesenchyme surrounding the umbilical vein and artery.

At 12.5 days pa, expression was more extensive than at 11.5 days pc.More of the nervous system exhibits expression of the transcript,including a larger portion of midbrain, the pontine areas, the basalganglia and the outermost layer of cortex. Neurons in this layer haveundergone mitosis in the subependymal layer of the cortex and migratedinto the mantle layer of the cerebral cortex as differentiated cells(Smart et al., J. Comp. Neurol. 116:325-347, 1961).

At 13.5 days pc, expression was seen throughout most of the brain. Theoutermost layer of the gut also appears to be expressing at this stage;these cells are neural crest derived and form the myenteric ganglia. At15.5 and 16.5 days pc, most of the neural crest derived neuralstructures have some expression. For example, the regions of the snoutthat will develop into the sensory structures at the base of thevibrissae, the pancreatic ganglia, the heart ganglion, the entericnervous system, and the sympathetic trunk all express the transcript.

There is no expression within the umbilicus at this stage. Twonon-neuronal structures express this gene, the gonad and the annulusfibrosus of the intervertebral disk. The olfactory bulb exhibits signalboth in the granule cells and within the tufted mitral cells. Within thenewborn brain, the transcript was expressed most extensively within thedifferentiating regions such as the septal area, olfactory bulb,inferior colliculus and hippocampus. In the adult brain, the gene wasexpressed in many areas including amygdala, cortex, hippocampus andthalamus. In the adult cerebellum the transcripts were detected in thePurkinje cell layer and in the deep cerebellar nuclei.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

Summary of Sequences

SEQ ID NO:1 is the nucleic acid sequence (and the deduced amino acidsequence) of cDNA encoding a novel human DS-CAM1 protein of the presentinvention.

SEQ ID NO:2 is the deduced amino acid sequence of a human DS-CAM1protein of the present invention.

SEQ ID NO:3 is the cDNA probe (labeled “E51”) used to isolate cDNAencoding human DS-CAM.

SEQ ID NO:4 is an MboI linker sequence.

SEQ ID NO:5 is a primer labeled B9-131F used in the RT-PCR assaydescribed in Example 5.

SEQ ID NO:6 is a primer labeled B9-131R used in the RT-PCR assaydescribed in Example 5.

SEQ ID NO:7 is the 5′ region of a partial mouse-derived cDNA cloneencoding an invention DS-CAM protein.

SEQ ID NO:8 is the middle region of a partial mouse-derived cDNA cloneencoding an invention DS-CAM protein.

SEQ ID NO:9 is the 3′ region of a partial mouse-derived cDNA cloneencoding an invention DS-CAM protein.

SEQ ID NO:10 is the nucleic acid sequence (and the deduced amino acidsequence) of cDNA encoding a novel human DS-CAM2 protein of the presentinvention.

SEQ ID NO:11 is the deduced amino acid sequence of a human DS-CAM2protein of the present invention, which is a splice variant of DS-CAM1(SEQ ID NO:2).

1-12. (canceled)
 13. An isolated protein comprising an animo acidsequence of SEQ ID NO: 2 or SEQ ID NO:
 11. 14. The protein according toclaim 13, further characterized by being expressed in a significantlyhigher amount in brain versus lung, liver or kidney.
 15. (canceled) 16.(canceled)
 17. An isolated protein having an amino acid sequence whichis encoded by a nucleotide sequence comprising substantially the samenucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO: 9, or SEQ ID NO:
 10. 18. (canceled)
 19. The isolatedprotein according to claim 17, wherein said protein is encoded by anucleotide sequence that comprises substantially the same nucleotidesequence as nucleotides 453-6185 set forth in SEQ ID NO: 1, ornucleotides 453-5168 set forth in SEQ ID NO:
 10. 20. (canceled)
 21. Anisolated antibody having specific reactivity with the protein accordingto claim
 13. 22. The antibody according to claim 21, wherein saidantibody is a monoclonal antibody.
 23. The antibody according to claim21, wherein said antibody is a polyclonal antibody. 24-27. (canceled)28. A method for identifying nucleic acids encoding a mammalian DS-CAMprotein, comprising: contacting a sample containing nucleic acids withan oligonucleotide consisting of at least 50 contiguous nucleotides of(a) the nucleotide sequence of SEQ ID NO: 1 to 10 or (b) the complementof the nucleotide sequence of (a), wherein said contacting is effectedunder high stringency hybridization conditions, and identifying thepresence of the nucleic acids which hybridize thereto.
 29. A method fordetecting the presence of a mammalian DS-CAM protein in a sample, saidmethod comprising contacting a test sample with an antibody according toclaim 21, detecting the presence of an antibody-DS-CAM complex, andtherefore detecting the presence of a mammalian DS-CAM in said testsample.
 30. (canceled)